PROJECT 1 SUMMARY/ABSTRACT Cancers that arise during childhood and adolescence are fundamentally different than cancers arising in adulthood. Harboring highly lineage specific oncogenic drivers, childhood cancers represent a diverse group of diseases due to misappropriation of normal human developmental processes. These cancers present few neoantigens, typically have a highly immunosuppressive tumor microenvironment, and we now have early data that the cell surface landscape (surfaceome) of these malignancies is unique and differs markedly from that of adult malignancies. Thus, here we propose to integrate genomics and proteomics to define and therapeutically exploit the surfaceome of high-risk pediatric malignancies. We hypothesize that optimal targets for antibody drug conjugate- (ADC) and chimeric antigen receptor (CAR) engineered T cell-based therapies will be lineage specific surface proteins that are not only abundantly expressed on the cell surface with limited expression in normal childhood tissues, but are also essential for tumor cell survival. We plan to test this hypothesis with three Specific Aims that while conceptually linear in nature for an individual immunotherapeutic target, will be approached in parallel. In Aim 1 we will discover the lineage-restricted cell surface proteins most abundantly and uniformly expressed in the plasma membrane of high-risk pediatric cancers using an integrative proteogenomic strategy. With Project 2, we will define intratumoral heterogeneity of expression for prioritized surfaceome proteins. Aim 2 will define the mechanisms by which candidate immunotherapeutic targets impart oncogenic dependencies on high-risk pediatric cancers, initially focusing on GPC2, PAPPA, and IL1RAP for detailed characterization, using genetic and pharmacologic interrogation strategies. Aim 3 will develop immunotherapeutic strategies, with the goal of showing potent efficacy in cellular and murine models of high- risk pediatric cancers, providing the preclinical rationale required for early phase clinical trials. This Project is directly responsive to RFA-CA-17-150, specifically the call for ?Identification of antigenic epitopes that are uniquely and abundantly expressed on childhood and adolescent cancers?. It is also highly integrated across the entire Center as all sites will focus on discovery and development of immunotherapeutic strategies in specific malignancies, but we will seek to define the subsets of patients most likely to respond in future trials by coordinated biomarker development for high risk childhood cancers. We will co-develop the multiplexed ion beam imaging strategy proposed in Project 2 to include lead candidate surfaceome proteins, here focused on intratumoral heterogeneity. Murine models developed in Project 3 will be used by Project 1 to study therapeutic engagement of the adaptive immune system. Taken together, we think that this Project within the overall proposed U54 Center, and the Pediatric Immunotherapy Discovery and Development Network (PI-DDN), will substantively impact childhood cancer outcomes by providing precision immunotherapy strategies based on unique tumor intrinsic vulnerabilities.